CN113943686B - Culture medium, culture method and application of culture of Pasteurella multocida EO630 strain - Google Patents
Culture medium, culture method and application of culture of Pasteurella multocida EO630 strain Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/102—Pasteurellales, e.g. Actinobacillus, Pasteurella; Haemophilus
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
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Abstract
The invention discloses a culture medium, a culture method and application of a culture of a swine pasteurella multocida EO630 strain, wherein the culture medium comprises the following components in percentage by mass: 2 to 3 percent of yeast extract powder, 2 to 3 percent of beef extract powder, 1 to 1.5 percent of peptone, 0.6 to 0.8 percent of glucose, 0.1 to 0.3 percent of sodium chloride, 0.2 to 0.5 percent of potassium dihydrogen phosphate, 0.5 to 0.8 percent of disodium hydrogen phosphate and the balance of water. The culture medium of the swine pasteurella multocida can realize ventilation fermentation culture of the swine pasteurella multocida under a proper environment, the fermentation culture time is short (only 14-16 h is needed), the viable count can reach 170-200 multiplied by 10 8 CFU/ml, and the culture medium has excellent industrial application value.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a culture medium, a culture method and application of a culture of a swine pasteurella multocida EO630 strain.
Background
The swine pasteurellosis (Swine pasteurellosis) is a sporadic infectious disease of swine caused by Pasteurella multocida, also called swine plague, laryngeal wind or "neck swelling disease", and mainly causes symptoms such as swine throat swelling, dyspnea, acute septicemia, pneumonia and the like. The disease is a common infectious disease of pigs, can occur all the year round, is parasitic to mice and other rodents, has pathogenicity to various animals and people, is most easy to infect pigs, has no obvious seasonal occurrence, but is more in wet and rainy condition, and causes such as malnutrition, long-distance transportation, change of feeding conditions, bad disease and the like to promote the occurrence of the disease, and is frequently and intensively developed. It is characterized in that most acute sepsis and pharyngitis are caused by laryngitis; acute type is cellulose pleuropneumonia; chronic forms are less common and mainly manifest chronic pneumonia.
The pasteurellosis vaccine plays an important role in treating swine pasteurellosis. The live bacteria for growing the pasteurella multocida on a large scale are key for preparing high-quality vaccines. At present, martin broth, modified Martin broth culture and the like are generally used for culturing Pasteurella multocida. The preparation process of the culture mediums is complex, comprises the procedures of meat purchasing, cold storage preservation, picking, mincing, heat preservation digestion, extraction and filtration and the like, and needs to consume a great deal of manpower and material resources, has a longer production period, is influenced by the quality of meat, and influences the stability of the quality of the culture mediums, thereby influencing the quality of vaccines. The reproductive capacity of the pasteurella multocida in pigs is related to the bacterial strain and the composition of a culture medium, and is also related to the culture conditions such as the culture temperature, the pH value, the culture time and the like. Therefore, the research on the growth and propagation culture medium suitable for the swine pasteurella multocida and the culture method thereof has very important practical significance.
Disclosure of Invention
In order to solve the problems, the invention provides a culture medium, a culture method and application of a culture of a swine pasteurella multocida EO630 strain, wherein the culture medium has short culture time and can promote high-yield viable count of the swine pasteurella multocida.
In order to achieve the aim, the invention provides a culture medium of a swine pasteurella multocida EO630 strain, which comprises the following components in percentage by mass: 2 to 3 percent of yeast extract powder, 2 to 3 percent of beef extract powder, 1 to 1.5 percent of peptone, 0.6 to 0.8 percent of glucose, 0.1 to 0.3 percent of sodium chloride, 0.2 to 0.5 percent of potassium dihydrogen phosphate, 0.5 to 0.8 percent of disodium hydrogen phosphate and the balance of water.
The preparation method of the culture medium comprises the following steps: weighing the components with the required amount according to the culture medium composition, uniformly mixing, adding a proper amount of water for injection, fully dissolving, adjusting the pH value to 7.5-7.7 by using a 2N sodium hydroxide solution, adding the water for injection to fix the volume, and sterilizing at 116 ℃ for 40min to obtain the culture medium.
The culture medium contains commercial biochemical reagents such as beef extract powder, yeast extract powder, peptone, glucose, sodium chloride, potassium dihydrogen phosphate and the like, has the advantages of convenient acquisition of culture medium components, simple and convenient process, stable quality, short production period, low manufacturing cost and the like.
Preferably, the medium comprises the following components in mass percent: 2.2 to 2.7 percent of yeast extract powder, 2.3 to 2.6 percent of beef extract powder, 1.2 to 1.4 percent of peptone, 0.7 to 0.75 percent of glucose, 0.15 to 0.25 percent of sodium chloride, 0.3 to 0.4 percent of potassium dihydrogen phosphate, 0.6 to 0.7 percent of disodium hydrogen phosphate and the balance of water.
The second aspect of the present invention provides a method for culturing a pasteurella multocida EO630 strain, the method comprising the steps of:
S1, diluting and inoculating a pasteurella multocida EO630 strain to a flat plate containing 0.1-0.2% of cracked whole blood and 3-5% of healthy animal serum, and culturing for 16-24 hours at 35-37 ℃ to obtain first-stage seeds of the pasteurella multocida EO630 strain; the plate is Martin agar plate;
S2, inoculating the first-level seeds of the porcine pasteurella multocida EO630 strain into a culture medium, and culturing for 18-20 hours at 36-37 ℃ to obtain the second-level seeds of the porcine pasteurella multocida EO630 strain;
S3, adding a culture medium into a fermentation tank according to 70-75 v/v percent, adding 0.1-0.2 v/v percent of defoamer according to the volume of the culture medium, sterilizing, reducing the temperature of the culture medium to 30-35 ℃, inoculating secondary seeds of the swine pasteurella multocida according to the inoculum size of 1-3 v/v percent, and culturing for 14-16 h at 36-37 ℃ and pH value of 7.2-7.5 to obtain the fermentation culture of the swine pasteurella multocida.
Wherein the culture medium is the culture medium.
The culture medium is used for realizing ventilation fermentation culture of the swine pasteurella multocida under a proper environment, and compared with the modified Martin soup culture medium (the culture time is 16-20 h), the fermentation culture time is short (only 14-16 h is needed), the viable count of the swine pasteurella multocida can reach 170-200 multiplied by 10 8 CFU/ml, and the swine pasteurella multocida has excellent industrial application value.
Preferably, in step S3, the pH of the medium is 7.0 to 7.8.
Preferably, in step S3, the cultivation is performed by a aeration cultivation method.
The third aspect of the invention provides the use of a fermentation culture obtained by culturing by the culture method described above for the preparation of a medicament or vaccine.
The pasteurella multocida liquid of the invention is prepared into vaccine, and can be used for preventing and treating related diseases caused by the pasteurella multocida.
Specifically, the fermentation culture is placed in a sealed manner at the temperature of 2-8 ℃ for 3-7 days for use.
And more specifically, the freeze-drying protective agent and the zymophyte liquid are mixed according to the proportion of 1 (6-8) and then freeze-dried to obtain the microbial inoculum.
Further, the freeze-drying protective agent is a mixture of gelatin and sucrose, and after mixing, the mass fraction of the gelatin is 1-2%, and the mass fraction of the sucrose is 4-6%.
Through the technical scheme, the invention has the following beneficial effects:
1. The invention improves the culture medium composition of the swine pasteurella multocida, and the culture medium contains commercial biochemical reagents such as beef extract powder, yeast extract powder, peptone, glucose, sodium chloride, potassium dihydrogen phosphate and the like, and has the advantages of convenient acquisition of the culture medium composition, simple and convenient process, stable quality, short production period, low manufacturing cost and the like.
2. The culture medium of the Pasteurella multocida can realize the aeration fermentation culture of the Pasteurella multocida in a proper environment, has short fermentation culture time (only 14-16 h is needed) compared with the culture medium of the improved Martin soup (the culture time is 16-20 h), has the viable count of 170-200 multiplied by 10 8 CFU/ml, and has excellent industrial application value.
3. The pasteurella multocida liquid can be prepared into vaccine, and can be used for preventing related diseases caused by the pasteurella multocida.
Detailed Description
The following describes specific embodiments of the present invention in detail with reference to examples. It should be understood that the detailed description and specific examples, while indicating and illustrating the invention, are not intended to limit the invention.
In the following examples, beef extract powder (LAB-LEMCO POWDER) was purchased from Thermo FISHER SCIENTIFIC, england under the trade designation LP0129; yeast extract powder was purchased from Hubei Angel Yeast Co., ltd., product number FM888; peptone was purchased from Hubei Angel Yeast Co., ltd., product number FP328; glucose was purchased from Shanghai national pharmaceutical Congress chemical company, cat# 10010518; sodium chloride is available from Shanghai national pharmaceutical group chemical company, inc., cat# 10019318; monopotassium phosphate is purchased from Shanghai national pharmaceutical group chemical reagent Co., ltd, cat# 10017518; disodium hydrogen phosphate dodecahydrate was purchased from Shanghai national pharmaceutical group chemical reagent Co., ltd, cat# 10010318; modified Martin broth dry powder media was purchased from Beijing aobo Co., ltd. Biotechnology, lot number 201816; the pasteurella multocida EO630 strain was identified, stored and supplied by the hapharmaceutical group biological vaccine limited.
Example 1
S1, uniformly mixing 2g of yeast extract powder, 2g of beef extract powder, 1g of peptone, 0.6g of glucose, 0.1g of sodium chloride, 0.2g of monopotassium phosphate and 0.5g of disodium hydrogen phosphate, adding a proper amount of water for injection, fully dissolving, adjusting the pH value to 7.5-7.7 by using a 2N sodium hydroxide solution, adding the water for injection to 100ml, and sterilizing at 116 ℃ for 40min to obtain the finished product.
S2, measuring the vacuum degree of an ampoule filled with the pasteurella multocida EO630 strain, opening the ampoule, inoculating the strain into the culture medium prepared in the step S1 containing 0.1% of lysed whole blood, culturing for 16 hours at 35-37 ℃, diluting the culture by a ten-fold serial dilution method, inoculating the properly diluted culture onto a modified Martin agar plate containing 0.1% of lysed whole blood and 3% of healthy animal serum, and culturing for 16 hours at 35-37 ℃.8 colonies with the same medium size are selected, streaked and inoculated on a plurality of blood agar slopes, cultured for 16 hours at the temperature of 35-37 ℃, and are used as first-stage seeds after being tested to be pure qualified. Preserving at 2-8 deg.c for no more than 14 days. The passage on the blood agar slope is not more than 5 generations.
S3, inoculating one first seed or one seed batch into a 5000ml serum bottle (filled with 3500ml of culture medium prepared in step S1) for culturing for 18h at 36-37 ℃, and obtaining the second seed of the swine Pasteurella multocida EO630 strain after inspection; the microscopic bacteria form accords with the standard of the veterinary biological product code, and the bacterial count reaches more than 8 hundred million/ml horsepower, namely the production seeds. Preserving at 2-8 deg.c for 72 hr.
S3, adding the culture medium prepared in the step S1 according to 70% (v/v) of the volume of a fermentation tank, adding 0.1% (v/v) of a defoaming agent according to the volume of the culture medium, sterilizing at 116 ℃ by high-pressure steam for 40min, cooling the culture medium to 30 ℃, inoculating the second-level seeds according to the inoculation amount of 1% (v/v), conducting aeration culture, and culturing for 14h at 36-37 ℃ and pH value of 7.2-7.5 to obtain the fermentation culture of the swine pasteurella multocida EO630 strain.
S4, sealing and placing the fermentation culture for 3 days at the temperature of 2-8 ℃, mixing the fermentation culture with a freeze-drying protective agent in a ratio of 6:1, and freeze-drying to obtain the vaccine.
Example 2
S1, uniformly mixing 3g of yeast extract powder, 3g of beef extract powder, 1.5g of peptone, 0.8g of glucose, 0.3g of sodium chloride, 0.5g of monopotassium phosphate and 0.8g of disodium hydrogen phosphate, adding a proper amount of water for injection, fully dissolving, adjusting the pH value to 7.5-7.7 by using a 2N sodium hydroxide solution, adding the water for injection to 100ml, and sterilizing at 116 ℃ for 40min to obtain the finished product.
S2, measuring the vacuum degree of an ampoule filled with the pasteurella multocida EO630 strain, opening the ampoule, inoculating the strain into the culture medium prepared in the step S1 containing 0.1% of lysed whole blood, culturing for 24 hours at 35-37 ℃, diluting the culture by a ten-fold serial dilution method, inoculating the properly diluted culture onto a modified Martin agar plate containing 0.2% of lysed whole blood and 5% of healthy animal serum, and culturing for 24 hours at 35-37 ℃. 10 colonies with medium size and consistent size are selected, streaked and inoculated on a plurality of blood agar slopes, cultured for 24 hours at the temperature of 35-37 ℃, and are used as first-stage seeds after being inspected to be purely qualified. Preserving at 2-8 deg.c for no more than 14 days. The passage on the blood agar slope is not more than 5 generations.
S3, inoculating one first seed or one seed batch into a 5000ml serum bottle (filled with 3500ml of culture medium) for culturing for 20 hours at 36-37 ℃, and obtaining the second seed of the swine pasteurella multocida EO630 strain after inspection; the microscopic bacteria form accords with the standard of the veterinary biological product code, and the bacterial count reaches more than 8 hundred million/ml horsepower, namely the production seeds. Preserving at 2-8 deg.c for 72 hr.
S3, adding the culture medium prepared in the step S1 according to 75% (v/v) of the volume of a fermentation tank, adding 0.2% (v/v) of a defoaming agent according to the volume of the culture medium, sterilizing at 116 ℃ by high-pressure steam for 40min, cooling the culture medium to 35 ℃, inoculating the second-level seeds according to the inoculation amount of 3% (v/v), conducting aeration culture, and conducting culture for 16h at 36-37 ℃ and pH value of 7.2-7.5 to obtain the fermentation culture of the swine Pasteurella multocida EO630 strain.
S4, sealing and placing the fermentation culture for 7 days at the temperature of 2-8 ℃, mixing the fermentation culture with a freeze-drying protective agent in a ratio of 8:1, and freeze-drying to obtain the vaccine.
Example 3
S1, uniformly mixing 2.2g of yeast extract powder, 2.3g of beef extract powder, 1.2g of peptone, 0.7g of glucose, 0.15g of sodium chloride, 0.3g of potassium dihydrogen phosphate and 0.6g of disodium hydrogen phosphate, adding a proper amount of water for injection, fully dissolving, adjusting the pH value to 7.5-7.7 by using a 2N sodium hydroxide solution, adding the water for injection to 100ml, and sterilizing at 116 ℃ for 40min to obtain the finished product.
S2, measuring the vacuum degree of an ampoule filled with the pasteurella multocida EO630 strain, opening the ampoule, inoculating the strain into the culture medium prepared in the step S1 containing 0.1% of lysed whole blood, culturing for 18 hours at 35-37 ℃, diluting the culture by a ten-fold serial dilution method, inoculating the properly diluted culture onto a modified Martin agar plate containing 0.1% of lysed whole blood and 4% of healthy animal serum, and culturing for 18 hours at 35-37 ℃. 9 colonies with medium size and uniform size are selected, streaked and inoculated on a plurality of blood agar slopes, cultured for 18 hours at the temperature of 35-37 ℃, and are used as first-stage seeds after being tested to be purely qualified. Preserving at 2-8 deg.c for no more than 14 days. The passage on the blood agar slope is not more than 5 generations.
S3, inoculating one first seed or one seed batch into a 5000ml serum bottle (filled with 3500ml of culture medium) for culturing for 19h at 36-37 ℃, and obtaining the second seed of the swine pasteurella multocida EO630 strain after inspection; the microscopic bacteria form accords with the standard of the veterinary biological product code, and the bacterial count reaches more than 8 hundred million/ml horsepower, namely the production seeds. Preserving at 2-8 deg.c for 72 hr.
S3, adding the culture medium prepared in the step S1 according to 70% (v/v) of the volume of a fermentation tank, adding 0.1% (v/v) of a defoaming agent according to the volume of the culture medium, sterilizing the culture medium by high-pressure steam at 116 ℃ for 40min, reducing the temperature of the culture medium to 33 ℃, inoculating the second-level seeds according to the inoculation amount of 1-3% (v/v), conducting aeration culture, and culturing for 15h at 36-37 ℃ and pH value of 7.2-7.5 to obtain the fermentation culture of the swine pasteurella multocida EO630 strain.
S4, sealing and placing the fermentation culture for 5 days at the temperature of 2-8 ℃, mixing the fermentation culture with a freeze-drying protective agent in a ratio of 7:1, and freeze-drying to obtain the vaccine.
Example 4
S1, uniformly mixing 2.7g of yeast extract powder, 2.6g of beef extract powder, 1.4g of peptone, 0.75g of glucose, 0.25g of sodium chloride, 0.4g of potassium dihydrogen phosphate and 0.7g of disodium hydrogen phosphate, adding a proper amount of water for injection, fully dissolving, adjusting the pH value to 7.5-7.7 by using a 2N sodium hydroxide solution, adding the water for injection to 100ml, and sterilizing at 116 ℃ for 40min to obtain the finished product.
S2, measuring the vacuum degree of an ampoule filled with the pasteurella multocida EO630 strain, opening the ampoule, inoculating the strain into the culture medium prepared in the step S1 containing 0.1% of lysed whole blood, culturing for 20 hours at 35-37 ℃, diluting the culture by a ten-fold serial dilution method, inoculating the properly diluted culture onto a modified Martin agar plate containing 0.2% of lysed whole blood and 5% of healthy animal serum, and culturing for 20 hours at 35-37 ℃. 10 colonies with medium size and consistent size are selected, streaked and inoculated on a plurality of blood agar slopes, cultured for 20 hours at the temperature of 35-37 ℃, and are used as first-stage seeds after being inspected to be purely qualified. Preserving at 2-8 deg.c for no more than 14 days. The passage on the blood agar slope is not more than 5 generations.
S3, inoculating one first seed or one seed batch into a 5000ml serum bottle (filled with 3500ml of culture medium) for culturing for 20 hours at 36-37 ℃, and obtaining the second seed of the swine pasteurella multocida EO630 strain after inspection; the microscopic bacteria form accords with the standard of the veterinary biological product code, and the bacterial count reaches more than 8 hundred million/ml horsepower, namely the production seeds. Preserving at 2-8 deg.c for 72 hr.
S3, adding the culture medium prepared in the step S1 according to 75% (v/v) of the volume of a fermentation tank, adding 0.2% (v/v) of a defoaming agent according to the volume of the culture medium, sterilizing at 116 ℃ by high-pressure steam for 40min, cooling the culture medium to 30 ℃, inoculating the second-level seeds according to the inoculation amount of 2% (v/v), conducting aeration culture, and conducting culture for 16h at 36-37 ℃ and pH value of 7.2-7.5 to obtain the fermentation culture of the swine Pasteurella multocida EO630 strain.
S4, sealing and placing the fermentation culture for 6 days at the temperature of 2-8 ℃, mixing the fermentation culture with a freeze-drying protective agent in a ratio of 8:1, and freeze-drying to obtain the vaccine.
Comparative example
S1, heating purified water to more than 80 ℃, weighing 100g of culture medium dry powder according to the preparation instructions of a purchased finished culture medium modified Martin broth dry powder culture medium (Beijing ao Boxing Biotechnology Co., ltd.), pouring the culture medium dry powder into a culture medium preparation barrel, heating the water to melt the culture medium dry powder, continuously stirring the culture medium dry powder in the water adding process, adding the water to 100ml, and properly prolonging the stirring time according to the melting degree of the culture medium dry powder until the culture medium dry powder is completely melted.
S2, measuring the vacuum degree of an ampoule filled with the pasteurella multocida EO630 strain, opening the ampoule, inoculating the strain into the culture medium prepared in the step S1 containing 0.1% of lysed whole blood, culturing for 20 hours at 35-37 ℃, diluting the culture by a ten-fold serial dilution method, inoculating the properly diluted culture onto a modified Martin agar plate containing 0.2% of lysed whole blood and 5% of healthy animal serum, and culturing for 20 hours at 35-37 ℃. 10 colonies with medium size and consistent size are selected, streaked and inoculated on a plurality of blood agar slopes, cultured for 20 hours at the temperature of 35-37 ℃, and are used as first-stage seeds after being inspected to be purely qualified. Preserving at 2-8 deg.c for no more than 14 days. The passage on the blood agar slope is not more than 5 generations.
S3, inoculating one first seed or one seed batch into a 5000ml serum bottle (3500 ml of S1 culture medium is filled) for culturing for 20 hours at 36-37 ℃, and obtaining the second seed of the swine Pasteurella multocida EO630 strain after inspection; the microscopic bacteria form accords with the standard of the veterinary biological product code, and the bacterial count reaches more than 8 hundred million/ml horsepower, namely the production seeds. Preserving at 2-8 deg.c for 48 hr.
S3, adding the culture medium prepared in the step S1 according to 75% (v/v) of the volume of a fermentation tank, adding 0.2% (v/v) of a defoaming agent according to the volume of the culture medium, sterilizing at 116 ℃ by high-pressure steam for 40min, cooling the culture medium to 30 ℃, inoculating the second-level seeds according to the inoculation amount of 2% (v/v), conducting aeration culture, and conducting culture for 20h at 36-37 ℃ and pH value of 7.2-7.5 to obtain the fermentation culture of the swine Pasteurella multocida EO630 strain.
S4, sealing and placing the fermentation culture for 6 days at the temperature of 2-8 ℃, mixing the fermentation culture with a freeze-drying protective agent in a ratio of 8:1, and freeze-drying to obtain the vaccine.
Performance detection
1. Detection of viable count of fermentation culture
The inspection method comprises the following steps: the fermentation cultures prepared in examples 1-4 and comparative example were counted in three annexes of the pharmacopoeia of the animal of the people's republic of China. Colony calculation and result judgment: and (3) visually observing the bacterial colonies, counting the number of the bacterial colonies on the bottom surfaces of the flat plates, and calculating the average bacterial colony number of the 3 flat plates, namely the total bacterial colony number contained in each milliliter of stock solution. If the colony grows in a sheet shape or the colony between different plates of the same dilution is different by more than 50%, the colony should be rechecked.
The number of viable bacteria in the fermentation cultures of the Pasteurella multocida pigs produced in examples 1 to 4 and comparative example, respectively, were 170×108CFU/ml、200×108CFU/ml、183×108CFU/ml、191×108CFU/ml、82×108CFU/ml.
2. Vaccine safety test
25 Healthy rabbits weighing 1.5-2kg are used, each group is divided into 5 groups, 5 vaccine 3 bottles of each group of the vaccine of the example and the comparative example are respectively taken and mixed, the vaccine is diluted into 10 parts per ml according to the parts of the bottle label, the skin (such as the inner side of thighs) of the injection site of the test rabbits is sterilized by 3% iodine, the iodine is removed by 75% alcohol cotton, and 1.0ml of the vaccine is injected into each rabbit by a 2.0ml syringe in a subcutaneous mode. The rabbits injected with the vaccines of examples 1 to 4 were alive and the rabbits injected with the vaccine of the comparative example were dead 1 after 10 days of observation.
3. Vaccine efficacy test
Efficacy test: either of the following methods is optional.
Test with mice: 16-18 g mice were 86 in total, and divided into 5 groups of 16 mice each, with 6 controls. Operation method and result judgment: the vaccine samples 1 bottle of example 1-example 4 and comparative example were taken, the vaccine was diluted to 0.2ml (1/30 head parts) in the bottle label, the skin of the injection site of the test mice (10) was sterilized with 75% alcohol cotton balls, and each subcutaneous injection was 0.2ml. After 14 days, 3 control mice under the same conditions were injected subcutaneously with 2MLD of Pasteurella multocida C44-8 strain (CVCC 44408) and 1MLD was injected subcutaneously with 3 control mice, and the calculation method for challenge was found in the standard procedure for bacterial preparation efficacy test attack deadly trap. The mice challenged with 2MLD were all killed, 2 mice challenged with 1MLD were killed, and 5 mice vaccinated with the vaccines of examples 1-4 survived with the vaccine of comparative example.
Test with rabbits: 1.5-2 kg rabbits are divided into 5 groups of 4 rabbits and 2 rabbits are compared, and the mass standard accords with the animal standards for production inspection of the animal pharmacopoeia of the people's republic of China. The rabbit raising management is carried out according to the experimental rabbit raising management program. Operation method and result judgment: sample 1 bottle was taken, the vaccine was diluted to 1/3 head/ml according to the bottle label, the skin of the injection site of the test rabbit (such as the inner thigh) was sterilized with 3% iodine, and the vaccine of example 1-example 4 and comparative example was injected into each group of rabbits, respectively, 1.0ml each, by deiodination with alcohol cotton. After 14 days of inoculation, 2 rabbits are inoculated together with the same condition, and each is subcutaneously injected with 80-100 CFU live bacteria of pasteurella multocida C44-8 strain (CVCC 44408), and the calculation method for the challenge is shown in the standard operation procedure of bacterial product efficacy test attack deadly trap. The control rabbits should all die after 10 days of observation, the immunized rabbits of the vaccines of examples 1-4 all survive, and 2 rabbits die after the vaccine of the comparative example is injected.
Inspection with pigs: healthy and susceptible pigs 28, which are about 20kg in weight after weaning for 1 month, are divided into 5 groups of 5 pigs each, and the other 2 pigs are used as controls. The animal quality standard accords with the animal standard for production inspection in the animal pharmacopoeia of the people's republic of China, and the pig raising management is carried out according to the inspection pig raising management program. Taking 1 bottle of vaccine sample of example 1-example 4 and comparative example, diluting the vaccine to 1 part/ml according to the bottle label, sterilizing the skin of the injection site of the test pig (such as the neck behind the ear) with 3% iodine, injecting 1.0ml subcutaneously each, inoculating 14 days later, and injecting 1MLD of pasteurella multocida C44-1 (CVCC 44401) virulent bacteria liquid subcutaneously in each pig together with 3 heads of the control pigs under the same condition, wherein the method for calculating the toxin is shown in the standard operation procedure of the test for efficacy of bacterial products, deadly trap. When the control pigs all died, the immunized pigs injected with the vaccines of examples 1 to 4 all survived, and the immunized pigs injected with the vaccines of the comparative examples died 1 head.
The preferred embodiments of the present invention have been described in detail above with reference to the examples, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solutions of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.
Moreover, any combination of the various embodiments of the invention can be made without departing from the spirit of the invention, which should also be considered as disclosed herein.
Claims (4)
1. The culture medium for the pasteurella multocida EO630 strain is characterized by comprising the following components in percentage by mass: 2.2-2.7% of yeast extract powder, 2.3-2.6% of beef extract powder, 1.2-1.4% of peptone, 0.7-0.75% of glucose, 0.15-0.25% of sodium chloride, 0.3-0.4% of potassium dihydrogen phosphate, 0.6-0.7% of disodium hydrogen phosphate and the balance of water.
2. A method for culturing a pasteurella multocida EO630 strain of pigs, comprising the steps of:
S1, diluting and inoculating a pasteurella multocida EO630 strain to a flat plate containing 0.1-0.2% of cracked whole blood and 3-5% of healthy animal serum, and culturing at 35-37 ℃ for 16-24 hours to obtain first-stage seeds of the pasteurella multocida EO630 strain;
s2, inoculating the primary seeds of the porcine pasteurella multocida EO630 strain into a culture medium, and culturing at 36-37 ℃ for 18-20 hours to obtain the secondary seeds of the porcine pasteurella multocida EO630 strain;
S3, adding a culture medium into a fermentation tank according to 70-75 v/v%, adding 0.1-0.2 v/v% of defoamer based on the volume of the culture medium, sterilizing, reducing the temperature of the culture medium to 30-35 ℃, inoculating secondary seeds of the swine pasteurella multocida EO630 strain according to the inoculum size of 1-3 v/v%, and culturing for 14-16 h at 36-37 ℃ and pH value of 7.2-7.5 to obtain a fermentation culture of the swine pasteurella multocida EO630 strain;
wherein the medium is the medium according to claim 1.
3. The method of claim 2, wherein in step S3, the pH of the culture medium is 7.0 to 7.8.
4. The method of culturing a strain of pasteurella multocida EO630 according to claim 2, characterized in that in step S3, the culture is carried out by aeration culture.
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以23号培养基制造猪肺疫氢氧化铝菌苗的研究;夏君明;中国兽医科技(12);第12页第(一)材料依据和培养基命名部分,第13-14页第(三)试验结果部分 * |
猪多杀性巴氏杆菌病活疫苗(EO630株)生产用合成培养基的研究;崔水保等人;中国兽药杂志;第48卷(第5期);摘要,第22页右栏实验方法部分,第24页左栏第3段 * |
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